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Effect of ATP on light responses of bovine ROS
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Abstracts labeled nucleotides and from a kinetic study of the light-scattering signal: the affinity for GDP ( ~ 10 6 M) is reduced in the R*-GTP binding protein complex to a value close to that of the affinity for GTP ( ~ 10 -s M); binding of GTP with this low affinity induces a transconformation of the protein resulting in a much higher affinity for GTP ( ~ 10-7 M). 1. Fung B. K. K. and Stryer L. Proc. natn. Acad. Sci. U.S.A. 77, 2500 (1980). 2. Kiihn H., Bennett N., Michel-Villaz M. and Chabre M. Proc. natn. Acad. Sci. U.S.A. 78, 6873 (1981). 3. Bennett N. Eur. J. Biochem. 123, 133 (1982).
LIPID SELECTIVITY IN THE BINDING OF PERIPHERAL PROTEINS TO ROD OUTER SEGMENTS N. RYBA a n d A. WATTS Department of Biochemistry, South Parks Road, Oxford We have investigated the binding of the light dependent GTPase (GBP) and PDE to ROS using lipid spin-labels which have the reporter group close to the membrane surface. The lipid labels show differential perturbations of the bilayer chain motion induced by GBP. In purified bovine disc membranes, sterol and phosphatidyl choline analogues are insensitive to the presence of extrinsic proteins while the fatty acid spin-label reported a marked apparent increase in lipid order in the presence of GBP. This change was saturated at physiological concentrations of GBP and insensitive to the bleached state of rhodopsin. The selectivity of the effects suggests that membrane binding may be specific for negatively charged lipids. The increase in order parameter can be explained either if GBP induces lateral phase separation around the binding surface, or if the protein withdraws associated lipids out of the membrane such that the label senses the more ordered environment near the polar-apolar interface.
LIGHT SCATTERING SIGNALS FROM ROS MEMBRANES R. UHL a n d H. DESEL MPI ffir biophysikalische Chemie, Am FaBberg, D-3400 G6ttingen, F.R.G. In recent years light scattering has been widely used for the study of rapid intracellular processes in photoreceptor cells. It has become obvious that there are several classes of light-induced light scattering transients in ROS, and these light scattering signals have been shown to originate from different structural domains. We have undertaken a systematic study of the light scattering behaviour of ROS membranes, using an apparatus that allows the simultaneous recording of light scattering transients at 8 different scattering angles. A major result of this examination has been the finding that the so called "Dissociation Signal" is not a reversal of the so called "Binding Signal" [1], earlier termed "P-Signal" by us, but a super-imposed signal that dominates at low scattering angles. At angles above 20° the "P- or Binding Signal" is still observed: when there is a "Dissociation Signal", however, the "P-Signal" exhibits a "Dissociation Signal"-like saturation behaviour. This and a number of other findings are discussed and interpreted in detail. 1. Kiihn H., Bennett N., Michel Villaz M. and Chabre M. Proc. natn. Acad. Sci. U.S.A. 78, 6873-6877 (1981).
EFFECT OF ATP ON LIGHT RESPONSES OF BOVINE ROS H. DESEL, T. BORYS, E. W. ABRAHAMSON a n d R. UHL MPI f/Jr biophysikalische Chemie, Am FaBberg, D-3400 Grttingen, F.R.G. ATP applied at millimolar concentrations to bovine ROS in the dark becomes hydrolysed and induces structural changes which can be monitored using light scattering techniques. We find that ATP-treatment
Abstracts influences the light-responses of ROS-membranes in three different ways: --light scattering transients observed without ATP are superimposed by another signal (Ai) of very high amplitude. AI corresponds to a decrease in light scattering. --The Metarhodopsin I/II reaction is accelerated by up to a factor of 10. --The maximum of the observed "extinction band" of metarhodopsin II is shifted by up to 10 nm to the blue, indicating a change in selective light scattering of the ROS membranes. The above effects are obtained from ROS with leaky plasma membrane as well as from preparations of intact disks stripped of soluble cytoplasmic and loosely membrane associated proteins. We have interpreted these results as being in support of a model whereby an ATPase located in the disk membrane is activated in the dark. This ATPase "energizes" the disk compartment by translocating protons into the intradiskal space.
GREATLY ENHANCED SENSITIVITY OF NEAR INFRARED LIGHT SCATTERING AS A PROBE OF ENZYMATIC PROCESSES IN ROS MEMBRANES E. A. DRATZ, J. W. LEWIS, J. L. MILLER a n d D. S. KLIGER University of Calif., Santa Cruz, CA 95064, U.S.A. Properties of the near infrared light scattering from ROS membrane suspensions are strongly dependent on membrane concentration and other factors. The transmission of near i.r. light is rapidly reduced by 25~ when >0.05~o of the rhodopsin is bleached at 5 mg/ml x 2 mm path. This transmission decrease requires GTP and is opposite in sign and 25 times larger in amplitude than a GTP dependent scattering signal previously reported in ROS [1]. The transmission returns to the preactive baseline after a time proportional to the amount of GTP added. Micromolar GMPPNP (a nonhydrolyzable GTP analogue) gives an irreversible light response. ATP strongly inhibits the return to the baseline of the GTP dependent signal. This effect of ATP is inhibited by the simultaneous presence of AMPPNP (a nonhydrolyzable ATP analogue) and so may require ATP hydrolysis. The amplitude of the light scattering signal and the velocity of activation of ROS phosphodiesterase saturate when >0.05~ of the rhodopsin is bleached. The large signal amplitude, strong membrane concentration dependence, and sedimentation experiments suggest that the nucleotide dependent light scattering signals described arise from changes in the state of membrane aggregation. The ATP effects are rationalized as due to rhodopsin phosphorylation adding negative charge to the membrane surface and keeping the membranes disaggregated. A second more intense flash produces increased light transmission. The latter signal is attributed to proton binding by bleached rhodopsin which decreases the negative charge repulsion and allows increased membrane aggregation. 1. Kuhn H. et al. Proc. nam. Acad. Sci. U.S.A. 78, 152 (1981).
PURIFICATION OF MULTIPLE FORMS OF RHODOPSIN KINASE ROBERT L. SOMERS a n d GERALD CHADER National Eye Institute, NIH, Bethesda, MD 20205, U.S.A. Photon absorption by rhodopsin causes it to undergo a series of conformational changes which are then somehow coupled to changes in the permeability of the plasma membrane. Among the reactions that occur subsequent to photon absorption is the phosphorylation of multiple serine and threonine residues at the C-terminal end of the rhodopsin molecule. This phosphorylation is catalyzed by the enzyme rhodopsin kinase (RK) which has strict substrate specificity for bleaching rhodopsin. Reports on the purification of RK have yielded conflicting values for its molecular weight; 52K [1] and 67K [2]. We have recently isolated two forms of R K both of which exhibit substrate specificity for bleaching rhodopsin but have different physical characteristics. The two forms of the kinase can be separated by either DEAE or Blue Sepharose chromatography. RK-I (52K) binds weakly to both DEAE and Blue Sepharose while RK-II (66K) binds more strongly and can be eluted with 0.1-0.15 M KC1. Both forms of the kinase have a low K,, for ATP as substrate, 8 and 4 # M for 52 and 66K respectively. The two forms differ however in that purified 66K kinase can be autophosphorylated by ATP while the 52K form can not. The possibility that the 52K form is a proteolytic product of 66K is argued against by two points: (1) PMSF addition throughout preparation of ROS and extracts does not alter the relative amount of 52K and (2) antibody to 52K does
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Abstracts labeled nucleotides and from a kinetic study of the light-scattering signal: the affinity for GDP ( ~ 10 6 M) is reduced in the R*-GTP binding protein complex to a value close to that of the affinity for GTP ( ~ 10 -s M); binding of GTP with this low affinity induces a transconformation of the protein resulting in a much higher affinity for GTP ( ~ 10-7 M). 1. Fung B. K. K. and Stryer L. Proc. natn. Acad. Sci. U.S.A. 77, 2500 (1980). 2. Kiihn H., Bennett N., Michel-Villaz M. and Chabre M. Proc. natn. Acad. Sci. U.S.A. 78, 6873 (1981). 3. Bennett N. Eur. J. Biochem. 123, 133 (1982).
LIPID SELECTIVITY IN THE BINDING OF PERIPHERAL PROTEINS TO ROD OUTER SEGMENTS N. RYBA a n d A. WATTS Department of Biochemistry, South Parks Road, Oxford We have investigated the binding of the light dependent GTPase (GBP) and PDE to ROS using lipid spin-labels which have the reporter group close to the membrane surface. The lipid labels show differential perturbations of the bilayer chain motion induced by GBP. In purified bovine disc membranes, sterol and phosphatidyl choline analogues are insensitive to the presence of extrinsic proteins while the fatty acid spin-label reported a marked apparent increase in lipid order in the presence of GBP. This change was saturated at physiological concentrations of GBP and insensitive to the bleached state of rhodopsin. The selectivity of the effects suggests that membrane binding may be specific for negatively charged lipids. The increase in order parameter can be explained either if GBP induces lateral phase separation around the binding surface, or if the protein withdraws associated lipids out of the membrane such that the label senses the more ordered environment near the polar-apolar interface.
LIGHT SCATTERING SIGNALS FROM ROS MEMBRANES R. UHL a n d H. DESEL MPI ffir biophysikalische Chemie, Am FaBberg, D-3400 G6ttingen, F.R.G. In recent years light scattering has been widely used for the study of rapid intracellular processes in photoreceptor cells. It has become obvious that there are several classes of light-induced light scattering transients in ROS, and these light scattering signals have been shown to originate from different structural domains. We have undertaken a systematic study of the light scattering behaviour of ROS membranes, using an apparatus that allows the simultaneous recording of light scattering transients at 8 different scattering angles. A major result of this examination has been the finding that the so called "Dissociation Signal" is not a reversal of the so called "Binding Signal" [1], earlier termed "P-Signal" by us, but a super-imposed signal that dominates at low scattering angles. At angles above 20° the "P- or Binding Signal" is still observed: when there is a "Dissociation Signal", however, the "P-Signal" exhibits a "Dissociation Signal"-like saturation behaviour. This and a number of other findings are discussed and interpreted in detail. 1. Kiihn H., Bennett N., Michel Villaz M. and Chabre M. Proc. natn. Acad. Sci. U.S.A. 78, 6873-6877 (1981).
EFFECT OF ATP ON LIGHT RESPONSES OF BOVINE ROS H. DESEL, T. BORYS, E. W. ABRAHAMSON a n d R. UHL MPI f/Jr biophysikalische Chemie, Am FaBberg, D-3400 Grttingen, F.R.G. ATP applied at millimolar concentrations to bovine ROS in the dark becomes hydrolysed and induces structural changes which can be monitored using light scattering techniques. We find that ATP-treatment
Abstracts influences the light-responses of ROS-membranes in three different ways: --light scattering transients observed without ATP are superimposed by another signal (Ai) of very high amplitude. AI corresponds to a decrease in light scattering. --The Metarhodopsin I/II reaction is accelerated by up to a factor of 10. --The maximum of the observed "extinction band" of metarhodopsin II is shifted by up to 10 nm to the blue, indicating a change in selective light scattering of the ROS membranes. The above effects are obtained from ROS with leaky plasma membrane as well as from preparations of intact disks stripped of soluble cytoplasmic and loosely membrane associated proteins. We have interpreted these results as being in support of a model whereby an ATPase located in the disk membrane is activated in the dark. This ATPase "energizes" the disk compartment by translocating protons into the intradiskal space.
GREATLY ENHANCED SENSITIVITY OF NEAR INFRARED LIGHT SCATTERING AS A PROBE OF ENZYMATIC PROCESSES IN ROS MEMBRANES E. A. DRATZ, J. W. LEWIS, J. L. MILLER a n d D. S. KLIGER University of Calif., Santa Cruz, CA 95064, U.S.A. Properties of the near infrared light scattering from ROS membrane suspensions are strongly dependent on membrane concentration and other factors. The transmission of near i.r. light is rapidly reduced by 25~ when >0.05~o of the rhodopsin is bleached at 5 mg/ml x 2 mm path. This transmission decrease requires GTP and is opposite in sign and 25 times larger in amplitude than a GTP dependent scattering signal previously reported in ROS [1]. The transmission returns to the preactive baseline after a time proportional to the amount of GTP added. Micromolar GMPPNP (a nonhydrolyzable GTP analogue) gives an irreversible light response. ATP strongly inhibits the return to the baseline of the GTP dependent signal. This effect of ATP is inhibited by the simultaneous presence of AMPPNP (a nonhydrolyzable ATP analogue) and so may require ATP hydrolysis. The amplitude of the light scattering signal and the velocity of activation of ROS phosphodiesterase saturate when >0.05~ of the rhodopsin is bleached. The large signal amplitude, strong membrane concentration dependence, and sedimentation experiments suggest that the nucleotide dependent light scattering signals described arise from changes in the state of membrane aggregation. The ATP effects are rationalized as due to rhodopsin phosphorylation adding negative charge to the membrane surface and keeping the membranes disaggregated. A second more intense flash produces increased light transmission. The latter signal is attributed to proton binding by bleached rhodopsin which decreases the negative charge repulsion and allows increased membrane aggregation. 1. Kuhn H. et al. Proc. nam. Acad. Sci. U.S.A. 78, 152 (1981).
PURIFICATION OF MULTIPLE FORMS OF RHODOPSIN KINASE ROBERT L. SOMERS a n d GERALD CHADER National Eye Institute, NIH, Bethesda, MD 20205, U.S.A. Photon absorption by rhodopsin causes it to undergo a series of conformational changes which are then somehow coupled to changes in the permeability of the plasma membrane. Among the reactions that occur subsequent to photon absorption is the phosphorylation of multiple serine and threonine residues at the C-terminal end of the rhodopsin molecule. This phosphorylation is catalyzed by the enzyme rhodopsin kinase (RK) which has strict substrate specificity for bleaching rhodopsin. Reports on the purification of RK have yielded conflicting values for its molecular weight; 52K [1] and 67K [2]. We have recently isolated two forms of R K both of which exhibit substrate specificity for bleaching rhodopsin but have different physical characteristics. The two forms of the kinase can be separated by either DEAE or Blue Sepharose chromatography. RK-I (52K) binds weakly to both DEAE and Blue Sepharose while RK-II (66K) binds more strongly and can be eluted with 0.1-0.15 M KC1. Both forms of the kinase have a low K,, for ATP as substrate, 8 and 4 # M for 52 and 66K respectively. The two forms differ however in that purified 66K kinase can be autophosphorylated by ATP while the 52K form can not. The possibility that the 52K form is a proteolytic product of 66K is argued against by two points: (1) PMSF addition throughout preparation of ROS and extracts does not alter the relative amount of 52K and (2) antibody to 52K does
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